The commonalities between human variant Creutzfeldt-Jakob Disease (vCJD), cervid chronic wasting disease (CWD) and sheep scrapie are quite remarkable. Hematogenous prions exhibiting association with the lymphoreticular system (LRS) are recapitulated in both human and animal transmissible spongiform encephalopathies (TSEs). In particular, vCJD, CWD and scrapie are all characterized by a long asymptomatic phase of disease from which infectious blood-borne prions efficiently transmit from one host to another. Because these TSEs traffic and disseminate similarly within susceptible hosts, the use of animal prion diseases provides a unique opportunity to evaluate the biology of blood-borne prions and their spread. The long-term goal of this research in my laboratory is to develop intervention strategies to detect, mitigate and prevent hematogenous prion spread. As a step towards this goal, we propose the use of animal TSEs to better define and understand the mechanisms associated with trafficking, dissemination and transmission of blood-borne prions. The use of animal TSEs will allow what has not- and cannot- be done with humans infected with vCJD, namely collect serial longitudinal blood samples from minutes after infection to terminal disease. The unifying hypothesis for this work is that blood-borne prions circulate within the host on/in specific cell phenotypes soon after infection and are responsible for early dissemination within the host and covert transmission between hosts. To test this hypothesis, we: 1) adapted fast, sensitive and cost effective in vitro conversion assays for the detection of blood-borne prions; 2) assembled a unique repository of longitudinal blood samples from natural and experimental TSE-infected cervids, rodents and primates; and 3) established facilities and collaborators with the capacity to house and assess TSEs in cervid and rodent in vivo models. We propose to address two specific aims, to 1) determine the temporal status of prionemia in TSE- infected hosts and 2) quantitatively determine which blood compartments and cell phenotypes harbor prions. We will employ blood, from naturally and experimentally infected animals (CWD-infected cervids, experimentally vCJD, BSE and CWD-infected primates and TME-infected hamsters) to investigate the efficacy of our recently established PrP conversion assay, whole blood real-time quaking induced conversion (wbRT- QuIC). The outcome of this work will establish the temporal status of prionemia as well as discern which compartments and specific cell phenotypes harbor prions. These results will be compared, where appropriate, to protein misfolding cyclic amplification and bioassay methods in place in my laboratory. These aims will provide what has long been sought in the prion field; elucidating the mechanisms of prion trafficking and dissemination throughout the host. This will permit timely detection, and thus, more effective therapies for human TSEs and other human protein misfolding diseases.